Mammalian Antibiotic Selection Options

Cloning: We currently sell five alternative mammalian antibiotic selection gene options. These have been inserted into four configurations within our plasmids to allow you to choose the selection system you would prefer. These include:

Under the direct control of the CMV promoter in the main MCS.

Under control of the PGK promoter immediately after the main multiple cloning site. The poly-adenylation signal is the same as that used by the upstream CMV promoter.

Under the control of an internal ribosome entry site (EMCV or FMDV). These can often demonstrate low, or variable activity depending on the sequence, cell line and sequence of the upstream gene. Yields of 1-5% of the upstream are not uncommon.

Under the control of either the Rous Sarcoma Virus (RSV) promoter (low expression) or the Human Ubiquitin (Ub) promoter (high expression), where the entire expression cassette is inserted away from the main multiple cloning site in the other half of our vector, flanked by two AscI restriction sites.

Mammalian Antibiotic Selection Gene Options:

We currently sell five different resistance genes, including:

Puromycin

Hygromycin

Blasticidin

Neomycin / G418

Zeocin

Please browse our resistance constructs using the buttons below. All of our constructs are designed for broad compatibility and versatile cloning, allowing you easily to produce any derivatives you require. As ever, if you prefer to outsource the cloning work, we are happy to undertake it rapidly and efficiently. Please use the 'Request Quote' button below to evaluate possible modifications to your chosen plasmids.

Puromycin Resistance Plasmid Information

Puromycin resistance features and uses:

Puromycin is an aminonucleoside antibiotic. It is produced naturally by the Streptomyces alboniger bacterium and disrupts peptide transfer on ribosomes causing premature chain termination of protein synthesis. It is a powerful inhibitor of translation in both prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene (pac), also from Streptomyces.

Puromycin selection: the principle

The pac gene is readily included into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of puromycin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum puromycin concentration, typically 1-10 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

Following transfection (24-48h) incubate cells in fresh medium containing Puromycin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.

Remove and replace the puromycin-containing medium every 3-4 days.

Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.

Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Our product range

Oxford Genetics provides a broad range of plasmids containingpuromycin resistance in a variety of configurations to meet all of your cloning requirements. The plasmid structure shown contains puromycin resistance driven by the ubiquitin promoter, with kanamycin for bacterial selection. The CMV promoter drives expression of any gene inserted into the MCS, and this plasmid also provides N terminal histidine and maltose binding protein tags, together with a TEV protease cleavage site to allow removal of both tags, if required, following protein production.

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the button below.

Hygromycin Resistance Plasmid Information

Hygromycin resistance features and uses:

Hygromycin B is an aminoglycoside antibiotic isolated from Streptomyces hygroscopicus. It disrupts translation by interfering with translocation and causes mistranslation at the 70S ribosome. Hygromycin B kills most bacteria, fungi and higher eukaryotes.
Resistance to hygromycin is conferred by the hph gene from E. coli.

Hygromycin B is normally used at concentrations of 50-200 µg/ml in eukaryotic cells.

Hygromycin selection: the principle

The 1 kb hph gene is readily include into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of hygromycin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum hygromycin concentration, typically 50-200 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

Following transfection (24-48h) incubate cells in fresh medium containing hygromycin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.

Remove and replace the hygromycin-containing medium every 3-4 days.

Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.

Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Our product range

The plasmid structure shown contains hygromycin resistance driven by the PGK promoter, with the MCS promoter positioned to drive your gene of interest (inserted into the MCS).

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the button below.

Blasticidin Resistance Plasmid Information

Blasticidin resistance features and uses:

Blasticidin is a nucleoside antibiotic produced by the bacterium Streptomyces griseochromogenes. It is a powerful inhibitor of translation in both prokaryotic and eukaryotic cells.

Resistance is conferred by the bsd gene from Aspergillus terreus.

Blasticidin acts quickly and causes cell death at low concentrations. E. coli strains are generally killed by concentrations of 50 µg/ml, while mammalian cells are killed by concentrations as low as 2 to 10 µg/ml.

Blasticidin selection: the principle

The 393 bp bsd gene encodes a polypeptide of 130 amino acids. It is readily included into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of blasticidin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum blasticidin concentration, typically 2-20 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

Following transfection (24-48h) incubate cells in fresh medium containing blasticidin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.

Remove and replace the blasticidin-containing medium every 3-4 days.

Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.

Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Our product range

Oxford Genetics provides a broad range of plasmids containingblasticidin resistance in a variety of configurations. The structure shown contains blasticidin resistance under control of the PGK promoter, allowing you to insert your gene of interest into the MCS under control of the CMV promoter. The plasmid also provides kanamycin resistance for selection in bacteria. Many diverse plasmids are available from our online catalogue, see buttons below.

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the Plasmid Builder button below.

Zeocin Resistance Plasmid Information

Zeocin resistance features and uses:

Zeocin is a copper chelated glycopeptide antibiotic related to bleomycin and causes cell death by intercalating into DNA and cleaving it. It is very toxic to a range of organisms, including mammalian and insect cells, as well as yeast, bacteria, and plants.

Resistance to Zeocin™ is conferred by the Sh ble gene, which binds and prevents it from interacting with DNA. The broad applicability of Zeocin resistance means that a single selectable marker can be used for several different cell types.

Zeocin selection: the principle

The 370 bp Sh ble gene is readily included into plasmids and provides a simple selectable marker, for many different cell types. For isolation of Zeocin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum Zeocin concentration, typically 25 - 400 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

Following transfection (24-48h) incubate cells in fresh medium containing Zeocin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.

Remove and replace the Zeocin-containing medium every 3-4 days.

Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.

Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Our product range

Oxford Genetics provides a broad range of plasmids containingZeocin resistance in a variety of configurations. The plasmid structure shown provides the Zeocin gene under regulatory control of the PGK promoter, with the CMV promoter available to drive expression of your gene of interest inserted into the MCS.

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the button below.